The presentation of exogenous foreign antigens to CD4-positive T-cells involves a complex series of events that includes the endocytosis of intact antigen, its proteolytic processing into peptides that then bind to newly synthesized MHC class II molecules, and the subsequent expression of the immunogenic class II-peptide complexes on the plasma membrane. While the general features of this pathway have been known for years, there is little precise information or agreement concerning the cell biological basis underlying each of these steps. Among the more critical issues remaining unresolved are the actual intracellular site(s) in which antigen processing and peptide-MHC complex formation occurs, whether accessory proteins exist that help select and/or transfer immunogenic peptides onto class II molecules, how proteins involved in antigen processing and presentation are targeted to their appropriate destinations, and whether the endocytic pathway in professional antigen- presenting cells is specialized to help mediate these events. Recently, strategies combining the techniques of cell biology and genetics with those of immunology have yielded significant new insights into class II- restricted antigen presentation. Specifically, it seems likely that at least some antigen-presenting cells contain a novel population of MHC class II vesicles (CIIV or MIIC) that serves as an important site for the formation of peptide<lass Il complexes. Distinct from conventional endosomes and lysosomes, CIIV are enriched in newly synthesized class Il molecules, receive extracellular antigen by endocytosis, and transiently accumulate peptide-loaded class 11 complexes prior to their appearance on the cell surface. Using free flow electrophoresis, the large scale isolation of highly purified CIIV is now possible, providing a unique opportunity to identify components involved in class Il-restricted antigen processing and biochemically dissect the events leading to the formation of immunogenic complexes. We propose to undertake just such a systematic analysis. First, we will analyze the composition of CIIV to identify known (e.g., HLA-DM) and novel components that may play a role in antigen processing. A new Ig-alpha-related protein that is a highly specific marker for this compartment has already been identified. Second, we will more precisely define the biogenesis and functional signifIcance of class Il compartments. Third, we will determine the extent to which specialized class Il compartments are expressed in professional and non- professional presenting cells. Of interest will be to determine whether CIIV are lymphocyte-specific, generally occurring, or formed as a result of class Il expression. Fourth, the sorting signals that specify the transport of CIIV components will be identified and characterized. Fifth, and most importantly, we will begin to establish assays to reconstitute stepwise the formation of immunogenic complexes in intact cells and in vitro, and to directly establish the role of CIIV and/or other compartments in these events.